Reaction Engineering

1. Reaction Engineering

2. Mass balance : depentend on reactor type -> S, P, X • Growth Kinetics: -> Monod model (substratedepleting model) -> Describeswhathappens in the reactor in steadystate(constantconditions) Model to describe what is going on in a Bio-reactor 1. Mass Ballance: In – Out + Reaction = Accumulation Biomass: FX0 - FX + ∫r dV = dn/dt dn/dt = d(XV)/dt r = dX/dt = µ X dn/dt=V (dX/dt) + X (dV/dt) 2. Monod Kinetics: 3. Steady state: dX/dt = 0 (NOT for Batch reactor!!!)

3. Fin = Fout ≠ 0 V = const. Continuous culture Control: • Concentration of a limiting nutrient • Dilution rate -> both influences X and P steady state = cell number, nutrient status remain constant -> Chemostat

5. Continuous culture: the chemostat 1. Concentration of a limiting nutrient 2. Dilution rate Results from a batch culture Monod Kinetics applies!!! D is dilution rate F is flow rate V is volume Substrate depletion kinetics !!

6. CV Mass Balance: In – Out + Reaction = Accumulation Math: FX0 - FX +  r V = dX/dt V Rearrange: F/V •(X0 –X) + r = dX/dt Chemostat: CSTR for Microbial Growth V = const. Fin = Fout ≠ 0 Output Growth

7. Chemostat: CFSTR for Microbial Growth Take limits as X and t  0 F/V •(X0 –X) + r = Substitute exponential growth equation for “r” Set X0 = 0 (no influent cells) Make steady state(SS) assumption (no net accumulation or depletion):  Let F/V = D = dilution rate Rearrange: D = m  

9. Cell Growth in Ideal Chemostat In Chemostat: µg=D, varying D obtains D~S Determination of Monod Parameters Washed out: If D is set at a value greater than µm (D > µm), the culture cannot reproduce quickly enough to maintain itself. µm = 0.2 hr-1 Chemostat technique: reliable, constant environment, operation may be difficult.

18. Fed batch fermentation -> In batch reactor, S and X are high. No transport of S or X and no control on µ. -> In chemostat, S and X are low. Transport of S or X and control on µ. -> In fed batch reactor. Substrate transport in, not out. No biomass transport. Why fed batch? • Low S  no toxicity / osmotic problem • High X  high P  easier downstream processing • Control of µ?

19. Fed batch fermentation Start feeding S0 S Feeding phase under substrate limited conditions S = 1 – 50 mg/l. Batch phase S0 5000 – 20000 mg/l time In substrate limited feeding phase, S is very low. Thus, one can use the pseudo steady state condition for substrate mass balance -> Useful for Antibiotic fermentation -> to overcome substrate inhibition!!

21. Mass Balance: In – Out + Reaction = Accumulation r = dX/dt = µ X Biomass: FX0 - FX + r V = dn/dt dn/dt = d(XV)/dt 0

22. substrate in substrate consumed Fed batch Substrate balance – no outflow (Fcout = 0), sterile feed St = SV and Xt = XV (mass of substrate or cells in reactor at a given time) S0 = substrate in feed stream Substrate balance no substrate out (Flow out = 0) Cell balance

23. Fed-batch Cell balance – sterile feed This can be a steady state reactor if substrate is consumed as fast as it enters (quasi-steady-state). Then dX/dt = 0 and μ = D, like in a chemostat. Recall, D = F / V

25. Fed batch What this means the total amount of cells in the reactor increases with time -> with increasing V dilution rate and μ decrease with time in fed batch culture Since μ = D, the growth rate is controlled by the dilution rate.

27. Minibioreactors -> Volumes below 100 ml Characterized by: -> area of application -> mass transfer -> mixing characteristics

28. Minibioreactors Why do we want to scale down ? - Parallelization (optimization, screening) • automatization • cost reduction What can you optimize? • Biocatalyst (organism) design • medium (growth conditions) design • process design

29. Minibioreactors • shake-flasks • microtiter plates • test tubes • stirred bioreactors • special reactors

30. Minibioreactors Shaking flasks: -> easy to handle -> low price -> volumne 25 ml – 5 L (filled with medium 20% of volumne) -> available with integrated sensors (O2, pH) -> limitation: O2 limitation (aeration) -> during growth improved by 1. baffled flasks 2. membranes instead of cotton -> during sampling

31. Minibioreactors Microtiter plates: -> large number of parallel + miniature reactors -> automation using robots -> 6, 12, 24, 48, 96, 384, 1536 well plates -> volumne from 25 μl – 5 ml -> integrated O2 sensor available Increased throughput rates allow applications: - screening for metabolites, drugs, new biocatalysts (enzymes) - cultivation of clone libraries - expression studies of recombinant clones - media optimization and strain development

32. Minibioreactors Microtiter plates: -> Problems: - O2 limitation (aeration) -> faster shaking -> contamination - cross-contamination - evaporation -> close with membranes - sampling (small volumne -> only micro analytical methods + stop shaking disturbs the respiration)

33. Minibioreactors Test tubes: -> useful for developing inoculums -> screening -> volumne 2 -25 ml (20% filled with medium) -> simple and low costs -> O2 transfer rate low -> usually no online monitoring (pH and O2) -> interruption of shaking during sampling

34. Minibioreactors Stirred Systems: -> homogeneous environment -> sampling, online monitoring, control possible without disturbance of culture -> increased mixing (stirring) + mass transfer (gassing rate)

35. Minibioreactors Stirred Systems – Stirred Minibioreactor -> T, pH, dissolved O2 can be controlled -> Volumne from 50 ml – 300 ml -> small medium requirenments -> low costs (isotope labeling) -> good for research -> good for continous cultivation -> Limitation: - system expensive due to minimization (control elements) - not good for high-throughput applications

36. Minibioreactors Stirred Systems – Spinner flask -> designed to grow animal cells -> high price instrument -> shaft containing a magnet for stirring -> shearing forces can be too big -> side arms for inoculation, sampling, medium inlet, outlet, ph probe, air (O2) inlet, air outlet -> continous reading of pH and O2 possible

37. Minibioreactors Special Devices – Cuvette based microreactor -> optical sensors (measuring online: pH, OD, O2) -> disposable -> volumne 4 ml -> air inlet/outlet -> magnet bead -> stirring -> similar performance as a 1 L batch reactor

38. Minibioreactors Special Devices – Miniature bioreactor with integrated membrane for MS measurement: -> custom made -> expensive -> a few ml -> online analysis of H2, CH4, O2, N2, CO2, and many other products, substrate,... -> used to follow respiratory dynamics of culture (isotope labeling) -> stirred vessel with control of T, O2, pH -> MS measurements within a few seconds to minutes -> continous detection -> fast kinetic measurements, metabolic studies

39. Minibioreactors Special Devices – Microbioreactor: -> Vessel 5 mm diameter round chamber -> Really small working volumne -> 5 μl -> integrated optical sensors for OD, O2, pH -> made out of polydimethylsiloxane (PDMS) -> transparent (optical measurements), permeable for gases (aeration) -> E. coli sucessfully grown -> batch and continous cultures possible -> similar profile as 500 ml batch reactors -> limitation: sampling (small volumne -> analytical methods !!!)

40. Minibioreactors NanoLiterBioReactor (NLBR): -> used for growing up to several 100 mammalien cells -> culture volumne around 20 μl -> online control of O2, pH, T -> culture chamber with inlet/outlet ports (microfluidic systems) -> manufactured by soft-lithography techniques -> made out of polydimethylsiloxane (PDMS) -> transparent (optical measurements), permeable for gases (aeration) -> direct monitoring of culture condition -> PDMS is transparent -> flourescence microscope -> limitation: batch culture very difficult-> too small volumne -> suffers from nutrient limitation -> But in principle system allows -> batch, fed-batch, continous

41. Minibioreactors NanoLiterBioReactor (NLBR): Circular with central post (CP-NBR) Chamber: 825 μm in diameter Volumne: 20 μl Perfusion Grid (PG-NBR) Similar Volumne Incorporated sieve With openings 3-8 μm -> small traps for cells Multi trap (MT-NBR) larger Volumne Incorporated sieve Opening similar -> multi trap system -> Seeding was necessary (Introduction of cells into chamber) -> 30 μm filtration necessary -> to prevent clogging in the chamber (aggregated cells) -> Flow rate of medium: 5-50 nl/min

42. Minibioreactors NanoLiterBioReactor (NLBR):

43. Minibioreactors NanoLiterBioReactor (NLBR):

45. Minibioreactors Why do we want micro-and nano reactors? Applications in: • Molecular biology • Biochemistry • Cell biology • Medical devices • Biosensors • > with the aim to look at single cells !!!

46. Minibioreactors Micro/Nanofluidic Device for Single cell based assay: -> used a microfluidic chip to capture passively a single cell and have nanoliter injection of a drug

47. Minibioreactors Micro/Nanofluidic Device for Single cell based assay: -> used a microfluidic chip to capture passively a single cell and have nanoliter injection of a drug Microchannel height: 20 μm (animal cells are smaller than 15 μm in diameter) -> If channel larger than 5 μm in diameter -> hydrophilic -> if channel smalles than 5 μm in diameter -> hydrophobic Gray area is hydrophobic -> air exchange possible -> no liquide (medium) can leak out

49. Class Exercise • Problem 6.17 • E. coli is cultivated in continuous culture under aerobic conditions with glucose limitation. When the system is operated at D= 0.2 hr-1, determine the effluent glucose and biomass concentrations assuming Monod kinetics (S0 = 5 g/l, mm= 0.25 hr-1 , KS = 100 mg/L, Y x/s = 0.4 g/g)